Spherical nucleic acids as a divergent platform for synthesizing RNA-nanoparticle conjugates through enzymatic ligation.
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ABSTRACT:
SUBMITTER: Rouge JL
PROVIDER: S-EPMC4174098 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chemically attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate entry. Here, we report studies aimed at determining the intracellular fate of SNAs and the trafficking events that occur inside C166 mouse endothelial cells after cellular entry. We show that SNAs traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation. Disassembly of oligonucleotides from the nanoparticle core is observed 16 h after cellular entry, most likely due to degradation by enzymes such as DNase II localized in late endosomes. Our observations point to these events being likely independent of core composition and treatment conditions, and they do not seem to be particularly dependent upon oligonucleotide sequence. Significantly and surprisingly, the SNAs do not enter the lysosomes under the conditions studied. To independently track the fate of the particle core and the fluorophore-labeled oligonucleotides that comprise its shell, we synthesized a novel class of quantum dot SNAs to determine that as the SNA structures are broken down over the 24 h time course of the experiment, the oligonucleotide fragments are recycled out of the cell while the nanoparticle core is not. This mechanistic insight points to the importance of designing and synthesizing next-generation SNAs that can bypass the degradation bottleneck imposed by their residency in late endosomes, and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome.
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Project description:Intracellular delivery of nucleic acids as gene regulation agents typically requires the use of cationic carriers or viral vectors, yet issues related to cellular toxicity or immune responses hamper their attractiveness as therapeutic candidates. The discovery that spherical nucleic acids (SNAs), polyanionic structures comprised of densely packed, highly oriented oligonucleotides covalently attached to the surface of nanoparticles, can effectively enter more than 50 different cell types presents a potential strategy for overcoming the limitations of conventional transfection agents. Unfortunately, little is known about the mechanism of endocytosis of SNAs, including the pathway of entry and specific proteins involved. Here, we demonstrate that the rapid cellular uptake kinetics and intracellular transport of SNAs stem from the arrangement of oligonucleotides into a 3D architecture, which supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. These results reinforce the notion that SNAs can serve as therapeutic payloads and targeting structures to engage biological pathways not readily accessible with linear oligonucleotides.
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Project description:Polyvalent oligonucleotide gold nanoparticle conjugates have unique fundamental properties including distance-dependent plasmon coupling, enhanced binding affinity, and the ability to enter cells and resist enzymatic degradation. Stability in the presence of enzymes is a key consideration for therapeutic uses; however the manner and mechanism by which such nanoparticles are able to resist enzymatic degradation is unknown. Here, we quantify the enhanced stability of polyvalent gold oligonucleotide nanoparticle conjugates with respect to enzyme-catalyzed hydrolysis of DNA and present evidence that the negatively charged surfaces of the nanoparticles and resultant high local salt concentrations are responsible for enhanced stability.
| S-EPMC3918421 | biostudies-literature
Project description:The sequence-dependent cellular uptake of spherical nucleic acid nanoparticle conjugates (SNAs) is investigated. This process occurs by interaction with class A scavenger receptors (SR-A) and caveolae-mediated endocytosis. It is known that linear poly(guanine) (poly G) is a natural ligand for SR-A, and it has been proposed that interaction of poly G with SR-A is dependent on the formation of G-quadruplexes. Since G-rich oligonucleotides are known to interact strongly with SR-A, it is hypothesized that SNAs with higher G contents would be able to enter cells in larger amounts than SNAs composed of other nucleotides, and as such, cellular internalization of SNAs is measured as a function of constituent oligonucleotide sequence. Indeed, SNAs with enriched G content show the highest cellular uptake. Using this hypothesis, a small molecule (camptothecin) is chemically conjugated with SNAs to create drug-SNA conjugates and it is observed that poly G SNAs deliver the most camptothecin to cells and have the highest cytotoxicity in cancer cells. Our data elucidate important design considerations for enhancing the intracellular delivery of spherical nucleic acids.
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Project description:SignificanceUsing SARS-CoV-2 as a relevant case study for infectious disease, we investigate the structure-function relationships that dictate antiviral spherical nucleic acid (SNA) vaccine efficacy. We show that the SNA architecture can be rapidly employed to target COVID-19 through incorporation of the receptor-binding domain, and that the resulting vaccine potently activates human cells in vitro and mice in vivo. Furthermore, when challenged with a lethal viral infection, only mice treated with the SNA vaccine survived. Taken together, this work underscores the importance of rational vaccine design for infectious disease to yield vaccines that elicit more potent immune responses to effectively fight disease.
| S-EPMC9168922 | biostudies-literature
Project description:Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many "undruggable" oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)-based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)--an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas--were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.
| S-EPMC4017940 | biostudies-literature
Project description:Lipid nanoparticle SNAs (LNP-SNAs) have been synthesized for the delivery of DNA and RNA to targets in the cytoplasm of cells. Both the composition of the LNP core and surface-presented DNA sequences contribute to LNP-SNA activity. G-rich sequences enhance the activity of LNP-SNAs compared to T-rich sequences. In the LNP core, increased cholesterol content leads to greater activity. Optimized LNP-SNA candidates reduce the siRNA concentration required to silence mRNA by 2 orders of magnitude compared to liposome-based SNAs. In addition, the LNP-SNA architectures alter biodistribution and efficacy profiles in mice. For example, mRNA within LNP-SNAs injected intravenously is primarily expressed in the spleen, while mRNA encapsulated by LNPs (no DNA on the surface) was expressed primarily in the liver with a relatively small amount in the spleen. These data show that the activity and biodistribution of LNP-SNA architectures are different from those of conventional liposomal SNAs and therefore potentially can be used to target tissues.
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Project description:Exosomes are a class of naturally occurring nanomaterials that play crucial roles in the protection and transport of endogenous macromolecules, such as microRNA and mRNA, over long distances. Intense effort is underway to exploit the use of exosomes to deliver synthetic therapeutics. Herein, transmission electron microscopy is used to show that when spherical nucleic acid (SNA) constructs are endocytosed into PC-3 prostate cancer cells, a small fraction of them (<1%) can be naturally sorted into exosomes. The exosome-encased SNAs are secreted into the extracellular environment from which they can be isolated and selectively re-introduced into the cell type from which they were derived. In the context of anti-miR21 experiments, the exosome-encased SNAs knockdown miR-21 target by approximately 50%. Similar knockdown of miR-21 by free SNAs requires a ?3000-fold higher concentration.
| S-EPMC3947239 | biostudies-literature
Project description:To understand the effect of three-dimensional oligonucleotide structure on protein corona formation, we studied the identity and quantity of human serum proteins that bind to spherical nucleic acid (SNA) nanoparticle conjugates. SNAs exhibit cellular uptake properties that are remarkably different from those of linear nucleic acids, which have been related to their interaction with certain classes of proteins. Through a proteomic analysis, this work shows that the protein binding properties of SNAs are sequence-specific and supports the conclusion that the oligonucleotide tertiary structure can significantly alter the chemical composition of the SNA protein corona. This knowledge will impact our understanding of how nucleic acid-based nanostructures, and SNAs in particular, function in complex biological milieu.
| S-EPMC4314381 | biostudies-literature
Project description:Topical application of nucleic acids offers many potential therapeutic advantages for suppressing genes in the skin, and potentially for systemic gene delivery. However, the epidermal barrier typically precludes entry of gene-suppressing therapy unless the barrier is disrupted. We now show that spherical nucleic acid nanoparticle conjugates (SNA-NCs), gold cores surrounded by a dense shell of highly oriented, covalently immobilized siRNA, freely penetrate almost 100% of keratinocytes in vitro, mouse skin, and human epidermis within hours after application. Significantly, these structures can be delivered in a commercial moisturizer or phosphate-buffered saline, and do not require barrier disruption or transfection agents, such as liposomes, peptides, or viruses. SNA-NCs targeting epidermal growth factor receptor (EGFR), an important gene for epidermal homeostasis, are > 100-fold more potent and suppress longer than siRNA delivered with commercial lipid agents in cultured keratinocytes. Topical delivery of 1.5 uM EGFR siRNA (50 nM SNA-NCs) for 3 wk to hairless mouse skin almost completely abolishes EGFR expression, suppresses downstream ERK phosphorylation, and reduces epidermal thickness by almost 40%. Similarly, EGFR mRNA in human skin equivalents is reduced by 52% after 60 h of treatment with 25 nM EGFR SNA-NCs. Treated skin shows no clinical or histological evidence of toxicity. No cytokine activation in mouse blood or tissue samples is observed, and after 3 wk of topical skin treatment, the SNA structures are virtually undetectable in internal organs. SNA conjugates may be promising agents for personalized, topically delivered gene therapy of cutaneous tumors, skin inflammation, and dominant negative genetic skin disorders.
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